(a) Structure of K163W Oraicryst from the I41 crystal form. The map (contoured at 1.3 σ) was calculated from 20 to 6.1 Å using native-sharpened amplitudes and experimental phases that were determined by MIRAS and were improved by 24-fold NCS averaging, solvent flattening and histogram matching (Materials and methods). The atomic model is shown in Cα representation (yellow). The crystal form is analogous to crystals of H206A Oraicryst and WT Oraicryst, and has analogous crystal packing. The root-mean-squared deviation (RMSD) for Cα positions between the structures of WT Oraicryst and K163W Oraicryst is 0.5 Å. (b) 4.35 Å resolution structure of K163W Oraicryst in the P42212 crystal form. Electron density (mesh) covering the channel is shown. The map (contoured at 1.3 σ) was calculated from 20 to 4.35 Å using sharpened amplitudes and phases that were determined by MR and were improved by threefold non-crystallographic symmetry (NCS) averaging, solvent flattening and histogram matching (Materials and methods). (c) Two opposing subunits of K163W Oraicryst (P42212 crystal form), showing the pore, with electron density from (b). Amino acids on the pore are depicted as sticks (conformations based on PDB 4HKS; Materials and methods). Anomalous-difference electron density in the pore is shown as magenta mesh (calculated from 30 to 8 Å resolution using anomalous differences as amplitudes, and contoured at 3.8 σ). Asterisks mark the locations of K163W substitutions. (d) Packing of K163W Oraicryst in the P42212 crystal form. The contents of each asymmetric unit (three Orai subunits) are colored a unique color. Two asymmetric units (e.g. blue and red) form a complete channel. The channels interact with one another in the crystal lattice via coiled-coil interactions between their M4-ext helices. (e) Anomalous-difference electron-density for heavy atom derivatives of K163W Oraicryst in the I41 space group. One channel of the asymmetric unit is depicted as ribbons. The map for the platinum (Pt) derivative (magenta mesh, calculated from 25 to 8.0 Å, and contoured at 4.5 σ) was calculated from data collected from a crystal soaked in PIP (Table 2), using anomalous differences as amplitudes and phases from (a). The analogous map for the mercury (Hg) derivative (green mesh, calculated from 25 to 8.0 Å, and contoured at 5 σ) was calculated from data collected from a crystal soaked in PCMB (Table 2). Each channel in the asymmetric unit has anomalous-difference density at these sites (24 sites for each derivative, Table 2). Cys215 and Met321 residues, to which the heavy atoms presumably bind, are depicted as sticks.